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Intracellular Glutathione (intracellular + glutathione)
Selected AbstractsIntracellular glutathione mediates the denitrosylation of protein nitrosothiols in the rat spinal cordJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2009Jorge M. Romero Abstract Protein S-nitrosothiols (PrSNOs) have been implicated in the pathophysiology of neuroinflammatory and neurodegenerative disorders. Although the metabolically instability of PrSNOs is well known, there is little understanding of the factors involved in the cleavage of S-NO linkage in intact cells. To address this issue, we conducted chase experiments in spinal cord slices incubated with S-nitrosoglutathione (GSNO). The results show that removal of GSNO leads to a rapid disappearance of PrSNOs (t½ , 2 hr), which is greatly accelerated when glutathione (GSH) levels are raised with the permeable analogue GSH ethyl ester. Moreover, PrSNOs are stable in the presence of the GSH depletor diethyl maleate, indicating that GSH is critical for protein denitrosylation. Inhibition of GSH-dependent enzymes (glutathione S-transferase, glutathione peroxidase, and glutaredoxin) and enzymes that could mediate denitrosylation (alcohol dehydrogense-III, thioredoxin and protein disulfide isomerase) do not alter the rate of PrSNO decomposition. These findings and the lack of protein glutathionylation during the chase indicate that most proteins are denitrosylated via rapid transnitrosylation with GSH. The differences in the denitrosylation rate of individual proteins suggest the existence of additional structural factors in this process. This study is relevant to our recent discovery that PrSNOs accumulate in the central nervous system of patients with multiple sclerosis. © 2008 Wiley-Liss, Inc. [source] Intracellular glutathione in stretch-induced cytokine release from alveolar type-2 like cellsRESPIROLOGY, Issue 1 2004Behrouz Jafari Objective: Ventilator-induced lung injury (VILI) is characterized by release of inflammatory cytokines, but the mechanisms are not well understood. We hypothesized that stretch-induced cytokine production is dependent on oxidant release and is regulated by intracellular glutathione (GSH) inhibition of nuclear factor ,B (NF-,B) and activator protein-1 (AP-1) binding. Methodology: Type 2-like alveolar epithelial cells (A549) were exposed to cyclic stretch at 15% strain for 4 h at 20 cycles/min with or without N-acetylcysteine (NAC) or glutathione monoethylester (GSH-e) to increase intracellular GSH, or buthionine sulfoximine (BSO), to deplete intracellular GSH. Results: Cyclic stretch initially caused a decline in intracellular GSH and a rise in the levels of isoprostane, a marker of oxidant injury. This was followed by a significant increase in intracellular GSH and a decrease in isoprostane. Stretch-induced IL-8 and IL-6 production were significantly inhibited when intracellular GSH was further increased by NAC or GSH-e (P < 0.0001). Stretch-induced IL-8 and IL-6 production were augmented when intracellular GSH was depleted by BSO (P < 0.0001). NAC blocked stretch-induced NF-,B and AP-1 binding and inhibited IL-8 mRNA expression. Conclusions: We conclude that oxidant release may play a role in lung cell stretch-induced cytokine release, and antioxidants, which increase intracellular GSH, may protect lung cells against stretch-induced injury. [source] Misregulation of gene expression in the redox-sensitive NF-,b-dependent limb outgrowth pathway by thalidomideDEVELOPMENTAL DYNAMICS, Issue 2 2002Jason M. Hansen Abstract Thalidomide is known to induce oxidative stress, but mechanisms have not been described through which oxidative stress could contribute to thalidomide-induced terata. Oxidative stress modulates intracellular glutathione (GSH) and redox status and can perturb redox-sensitive processes, such as transcription factor activation and/or binding. Nuclear factor-kappa B (NF-,B), a redox-sensitive transcription factor involved in limb outgrowth, may be modulated by thalidomide-induced redox shifts. Thalidomide-resistant Sprague-Dawley rat embryos (gestation day [GD] 13) treated with thalidomide in utero showed no changes in GSH distribution in the limb but thalidomide-sensitive New Zealand White rabbit embryos (GD 12) showed selective GSH depletion in the limb bud progress zone (PZ). NF-,B and regulatory genes that initiate and maintain limb outgrowth and development, such as Twist and Fgf-10, are selectively expressed in the PZ. Green fluorescent protein (GFP) reporter vectors containing NF-,B binding promoter sites were transfected into both rat and rabbit limb bud cells (LBCs). Treatment with thalidomide caused a preferential decrease in GFP expression in rabbit LBCs but not in rat LBCs. N-acetylcysteine and ,-N-t-phenylbutyl nitrone (PBN), a free radical trapping agent, rescued GFP expression in thalidomide-treated cultures compared with cultures that received thalidomide only. In situ hybridization showed a preferential decrease in Twist, Fgf-8, and Fgf-10 expression after thalidomide treatment (400 mg/kg per day) in rabbit embryos. Expression in rat embryos was not affected. Intravenous cotreatment with PBN and thalidomide (gavage) in rabbits restored normal patterns and localization of Twist, Fgf-8, and Fgf-10 expression. These findings show that NF-,B binding is diminished due to selective thalidomide-induced redox changes in the rabbit, resulting in the significant attenuation of expression of genes necessary for limb outgrowth. © 2002 Wiley-Liss, Inc. [source] Phase II study of arsenic trioxide and ascorbic acid for relapsed or refractory lymphoid malignancies: a Wisconsin Oncology Network study,HEMATOLOGICAL ONCOLOGY, Issue 1 2009JE Chang Abstract Arsenic trioxide (As2O3) has established clinical activity in acute promyelocytic leukaemia and has pre-clinical data suggesting activity in lymphoid malignancies. Cell death from As2O3 may be the result of oxidative stress. Agents which deplete intracellular glutathione, such as ascorbic acid (AA), may potentiate arsenic-mediated apoptosis. This multi-institution phase II study investigated a novel dosing schedule of As2O3 and AA in patients with relapsed or refractory lymphoid malignancies. Patients received As2O3 0.25,mg/kg IV and AA 1000,mg IV for five consecutive days during the first week of each cycle followed by twice weekly infusions during weeks 2,6. Cycles were repeated every 8 weeks. The primary end point was objective response. In a subset of patients, sequential levels of intracellular glutathione and measures of Bcl-2 and Bax gene expression were evaluated in peripheral blood mononuclear cells during treatment. Seventeen patients were enrolled between March 2002 and February 2004. The median age was 71, and the majority of enrolled patients had non-Hodgkin's lymphoma (12/17). Sixteen patients were evaluable, and one patient with mantle cell lymphoma achieved an unconfirmed complete response after five cycles of therapy for an overall response rate of 6%. The trial, which had been designed as a two-stage study, was closed after the first stage analysis due to lack of activity. Haematologic toxicities were the most commonly reported events in this heavily pre-treated population, and comprised the majority of grade 3 and 4 toxicities. Intracellular depletion of glutathione was not consistently observed during treatment. As2O3 and AA in this novel dosing strategy was generally well tolerated but had limited activity in patients with relapsed and refractory lymphoid malignancies. Copyright © 2008 John Wiley & Sons, Ltd. [source] Glutamate is a determinant of cellular proliferation through modulation of nuclear factor E2 p45-related factor-2 expression in osteoblastic MC3T3-E1 cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2007Kyosuke Uno Activation of particular glutamate (Glu) receptors is shown to promote cellular differentiation toward maturation during osteoblastogenesis. In the present study, we have evaluated the possible modulation by Glu of cellular proliferation in osteoblastic cells endowed to proliferate for self-renewal and to differentiate toward matured osteoblasts. Exposure to Glu significantly suppressed the proliferation activity at a concentration over 500 µM without inducing cell death in osteoblastic MC3T3-E1 cells before differentiation. The suppression by Glu occurred in a manner sensitive to the prevention by either cystine or reduced glutathione. Expression of mRNA was for the first time shown with the cystine/Glu antiporter composed of xCT and 4F2hc subunits in these undifferentiated osteoblastic cells. A significant decrease was seen in intracellular total glutathione levels in undifferentiated MC3T3-E1 cells cultured with Glu, indeed, whereas the cellular proliferation activity was drastically decreased by the addition of the glutathione depleter cyclohexene-1-one and the glutathione biosynthesis inhibitor L -buthionine-[S,R]-sulfoximine, respectively. Exposure to Glu led to a significant increase in mRNA expression of nuclear factor E2 p45-related factor 2 (Nrf2) together with the generation of reactive oxygen species, while a significant decrease was seen in the proliferation activity in MC3T3-E1 cells with stable overexpression of Nrf2. These results suggest that Glu could suppress the cellular proliferation toward self-renewal through a mechanism associated with the upregulation of Nrf2 expression in association with the depletion of intracellular glutathione after promoting the retrograde operation of the cystine/Glu antiporter in undifferentiated MC3T3-E1 cells. J. Cell. Physiol. 213: 105,114, 2007. © 2007 Wiley-Liss, Inc. [source] HIV-Tat protein induces oxidative and inflammatory pathways in brain endotheliumJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Michal Toborek Abstract Impaired function of the brain vasculature might contribute to the development of HIV-associated dementia. For example, injury or dysfunction of brain microvascular endothelial cells (BMEC) can lead to the breakdown of the blood,brain barrier (BBB) and thus allow accelerated entry of the HIV-1 virus into the CNS. Mechanisms of injury to BMEC during HIV-1 infection are not fully understood, but the viral gene product Tat may be, at least in part, responsible for this effect. Tat can be released from infected perivascular macrophages in the CNS of patients with AIDS, and thus BMEC can be directly exposed to high concentrations of this protein. To study oxidative and inflammatory mechanisms associated with Tat-induced toxicity, BMEC were exposed to increasing doses of Tat1,72, and markers of oxidative stress, as well as redox-responsive transcription factors such as nuclear factor-,B (NF-,B) and activator protein-1 (AP-1), were measured. Tat1,72 treatment markedly increased cellular oxidative stress, decreased levels of intracellular glutathione and activated DNA binding activity and transactivation of NF-,B and AP-1. To determine if Tat1,72 can stimulate inflammatory responses in brain endothelium in vivo, expression of monocyte chemoattractant protein-1 (MCP-1), an NF-,B and AP-1-dependent chemokine, was studied in brain tissue in mice injected with Tat1,72 into the right hippocampus. Tat1,72 markedly elevated the MCP-1 mRNA levels in brain tissue. In addition, a double immunohistochemistry study revealed that MCP-1 protein was markedly overexpressed on brain vascular endothelium. These data indicate that Tat1,72 can induce redox-related inflammatory responses both in in vitro and in vivo environments. These changes can directly lead to disruption of the BBB. Thus, Tat can play an important role in the development of detrimental vascular changes in the brains of HIV-infected patients. [source] Investigation of Protective Reactions Against Cadmium Toxicity in the Cells Established from a Transgenic Mouse Deficient in the Metallothionein GenesJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 2 2003Tetsuya Abe Objective:, To characterize a fibroblast cell strain which we established from an metallothionein (MT) knock-out (KO) mouse and to determine whether expression of the Hsp genes induced by cadmium is related to expression of the MT-I and -II genes. Methods:, We established a fibroblast cell strain (named "MT-KO2") derived from the peritoneum of an MT-KO mouse which is deficient in the MT-I and -II genes. We determined an expression of MT-I, Hsp32 and Grp 78 genes by Northern blot analysis. Results:, The mRNA level of MT-I, an isoform of the MT gene products, was induced dose-dependently in responce to increasing concentrations of CdCl2 (5,25 µM) in a fibroblast cell strain derived from the peritoneum of an MT wild type mouse (named "MT-W3"). But it was not induced in MT-KO2 cells after the same treatment. There was no significant difference between MT-KO2 and MT-W3 cells in a concentration of intracellular glutathione (reduced form) under normal conditions. MT-KO2 cells were not more sensitive to cytotoxicity of CdCl2 than in MT-W3 cells. Expression of the Hsp32 gene was more extensively enhanced in MT-KO2 cells than in MT-W3 cells after treatment with 5,10 µM CdCl2 for 5 hours. Furthermore, the cellular concentration of reduced glatathione (GSH) was also more increased in MT-KO2 cells than in MT-W3 cells after treatment with 50 µM CdCl2 for 3 hours. Conclusions:, Expression of the Hsp32 gene tends to be increased in MT-KO2 cells in response to cadmium exposure. The expression of the Hsp32 gene and increase in the cellular concentration of GSH may be augmented to compensate for the impaired expression of the MT genes in MT-KO2 cells. [source] Alcohol-Induced Endothelial Changes Are Associated With Oxidative Stress and Are Rapidly Reversed After WithdrawalALCOHOLISM, Issue 10 2005Giorgio Soardo Abstract: Background: Although heavy alcohol drinkers are at an increased risk of developing cardiovascular events, moderate alcohol intake is associated with reduced incidence of cardiovascular death. This paradox might reflect a dose-related effect of different alcohol intakes on endothelial function and this, in turn, might depend on changes in oxidative stress Methods: We tested the effects of alcohol withdrawal in heavy alcohol consumers and compared the plasma levels of endothelin-1, nitric oxide, plasminogen activator inhibitor-1, von Willebrand factor, malondialdehyde, and intracellular glutathione with those of alcoholics that did not modify their alcohol intake and teetotalers. In human endothelial cells that had been cultured for 2 weeks in the presence of different concentrations of ethanol, we assessed the same parameters after withdrawal of ethanol exposure Results: Alcohol increased the levels of endothelin-1, nitric oxide, and plasminogen activator inhibitor-1 and decreased the levels of von Willebrand factor both in vivo and in vitro. These changes were dose dependent, rapidly reversed after withdrawal of exposure, and associated with the presence of increased oxidative stress as indicated by increased levels of both malondialdehyde and intracellular glutathione. Blockade of oxidative stress by incubation of endothelial cells in the presence of oxidants' scavengers prevented the alcohol-induced functional modifications of the endothelium Conclusions: Alcohol affects endothelial function with an effect that is mediated by an activated oxidative stress and is rapidly reversed after withdrawal. Dose-related endothelial responses to different alcohol intakes might translate in either vascular protection or vascular damage. [source] Regulation of type I plasminogen activator inhibitor in human gingival fibroblasts with cyclosporine AORAL DISEASES, Issue 4 2010Y-C Ho Oral Diseases (2010) 16, 396,401 Objectives:, Cyclosporine A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of gingival overgrowth. Type I plasminogen activator inhibitor (PAI-1) has shown to play an important role in CsA-induced gingival overgrowth. However, little is known about whether factors can modulate CsA-induced PAI-1 expression. Methods:, Cytotoxicity, reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay were used to investigate the effects of Human gingival fibroblasts (HGFs) exposed to CsA. In addition, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, interlukin-1,, tumor necrosis factor-,, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, signal-regulated protein kinase (ERK) inhibitor PD98059 and cell-permeable glutathione precursor N -acetyl- L -cysteine (NAC) were added to test how they modulated the effects of CsA-induced PAI-1 expression. Results:, The concentration of CsA higher than 500 ng ml,1 demonstrated cytotoxicity to HGFs (P < 0.05). Periodontal pathogens as well as proinflammatory cytokines were found to increase the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). Pharmacological agents NAC, U0126, and PD98059 were found to decrease the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). Conclusions:, Cyclosporine A (CsA) may predispose to gingival overgrowth under inflammatory environments. The regulation of PAI-1 expression induced by CsA might be critically related with the intracellular glutathione and the ERK-MAPK pathway. [source] Neuroprotective effects of 3,5-dicaffeoylquinic acid on hydrogen peroxide-induced cell death in SH-SY5Y cellsPHYTOTHERAPY RESEARCH, Issue 3 2005Sung-Soo Kim Abstract Oxidative stress plays an important role in the pathological processes of a variety of neurodegenerative diseases. The neuroprotective effects of 3,5-diCQA and 3,4-diCQA, two caffeoylquinic acid derivatives present in Dipsacus asper, on hydrogen peroxide (H2O2)-induced neuronal cell damage were evaluated in this study. SH-SY5Y cells treated with H2O2 exhibited a decrease in survival and intracellular glutathione and also an increase in the caspase-3 activity. However, pretreatment of cells with 3,5-diCQA attenuated the neuronal death and caspase-3 activation induced by H2O2. In addition, 3,5-diCQA restored H2O2 -induced depletion of intracellular glutathione. 3,5-diCQA showed significant protective effects although it could not completely suppress H2O2 -induced cell injury to control levels. The data suggest that 3,5-diCQA might be a potential therapeutic agent for treating or preventing neurodegenerative diseases implicated with oxidative stress. Copyright © 2005 John Wiley & Sons, Ltd. [source] Intracellular glutathione in stretch-induced cytokine release from alveolar type-2 like cellsRESPIROLOGY, Issue 1 2004Behrouz Jafari Objective: Ventilator-induced lung injury (VILI) is characterized by release of inflammatory cytokines, but the mechanisms are not well understood. We hypothesized that stretch-induced cytokine production is dependent on oxidant release and is regulated by intracellular glutathione (GSH) inhibition of nuclear factor ,B (NF-,B) and activator protein-1 (AP-1) binding. Methodology: Type 2-like alveolar epithelial cells (A549) were exposed to cyclic stretch at 15% strain for 4 h at 20 cycles/min with or without N-acetylcysteine (NAC) or glutathione monoethylester (GSH-e) to increase intracellular GSH, or buthionine sulfoximine (BSO), to deplete intracellular GSH. Results: Cyclic stretch initially caused a decline in intracellular GSH and a rise in the levels of isoprostane, a marker of oxidant injury. This was followed by a significant increase in intracellular GSH and a decrease in isoprostane. Stretch-induced IL-8 and IL-6 production were significantly inhibited when intracellular GSH was further increased by NAC or GSH-e (P < 0.0001). Stretch-induced IL-8 and IL-6 production were augmented when intracellular GSH was depleted by BSO (P < 0.0001). NAC blocked stretch-induced NF-,B and AP-1 binding and inhibited IL-8 mRNA expression. Conclusions: We conclude that oxidant release may play a role in lung cell stretch-induced cytokine release, and antioxidants, which increase intracellular GSH, may protect lung cells against stretch-induced injury. [source] Docetaxel enhances the cytotoxicity of cisplatin to gastric cancer cells by modification of intracellular platinum metabolismCANCER SCIENCE, Issue 8 2004Shingo Maeda We have examined the combined anticancer effects of docetaxel (DOC) and cisplatin (CDDP) in vitro using the gastric cancer cell lines MKN-45, MKN-74, and TMK-1. Treatment of the cell lines with 30 ,g/ml of DOC for 24 h followed by incubation with 3 or 10 ,g/ml of CDDP for 24 h showed a clear synergistic effect. Sequence dependency of the agents was observed in these cell lines: DOC followed by CDDP (DC) showed a stronger antitumor effect than CDDP followed by DOC (CD) in all cell lines. To clarify the mechanism of action of the DC combination, total intracellular platinum (Pt) levels were evaluated after treatment with CDDP alone or combined with DC. For the MKN-45 and -74 cell lines, cells treated with DOC (10 ,g/ml for 12 h) and then CDDP showed significantly increased intracellular Pt accumulation compared to cells treated with CDDP alone. We also investigated alterations in intracellular glutathione (GSH) concentration in response to DOC and CDDP. MKN-45 and -74 cells pretreated with DOC (10 ,g/ml for 12 h) showed significantly increased intracellular GSH levels compared to cells administered CDDP only. To explain these findings, messenger RNA (mRNA) levels for multidrug resistance-associated protein-1 (MRP-1), the ATP-dependent pump for Pt-GSH complexes, were quantified in CDDP-treated MKN-45 cells with and without DOC pretreatment. While CDDP administration increased MRP-1 mRNA expression in MKN-45 cells, MRP-1 was not up-regulated after CDDP administration in DOC pretreated MKN-45 cells. Our results suggested that the enhanced CDDP toxicity due to DOC pretreatment may be related to the accumulation of intracellular Pt-GSH complexes, because DOC appears to suppress the MRP-1 up-regulation induced by CDDP exposure in gastric cancer cells. [source] Inhibitory effects of N -acetylcysteine on the functional responses of human eosinophils in vitroCLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2007M. Martinez-Losa Summary Background Oxidative stress appears to be relevant in the pathogenesis of inflammation in allergic diseases like bronchial asthma. Eosinophils are oxidant-sensitive cells considered as key effectors in allergic inflammation. Objective The aim of this work was to study the effects of the clinically used antioxidant N -acetyl- l -cysteine (NAC) on the functional responses of human-isolated eosinophils. Methods Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of NAC were investigated on the generation of reactive oxygen species (chemiluminescence and flow cytometry), Ca2+ signal (fluorimetry), intracellular glutathione (GSH; flow cytometry), p47phox,p67phox translocation (Western blot) and eosinophil cationic protein (ECP) release (radioimmunoassay). Results NAC (0.1,1 mm) inhibited the extracellular generation of oxygen species induced by N -formyl- l -methionyl- l -leucyl- l -phenylalanine (fMLP) and eotaxin (in the presence of IL-5) with ,logIC50 values of 3.61±0.03 and 3.36±0.09, respectively. Also, the intracellular generation of hydrogen peroxide was virtually abolished by NAC (0.5,1 mm). NAC (1 mm) did not alter the fMLP-induced Ca2+ signal but augmented the eosinophil content of reduced GSH and inhibited p47phox,p67phox translocation. NAC inhibited the release of ECP (,90% inhibition at 1 mm) from fMLP-activated eosinophils. Conclusion Inhibition by NAC of human eosinophil functions in vitro is potentially useful in the treatment of allergic inflammation. [source] | |||||||||||||